Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Abstract

Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra-stable consensus-designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch-like ECH-associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1-binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.

Engineering mono- and multi-valent inhibitors on a modular scaffold

“…Our condence in the broad applicability of the scaffold to the display of diverse SLiMs arises from the nding that we could effectively gra two SLiMs with very different binding conformations onto the CTPR loops. The Keap1-binding peptide from Nrf2 adopts a tight turn-like conformation, 33 whereas the TBP in this work binds to ARC domains in a highly extended conformation. 36,55 It might appear that a limiting factor in this multivalent platform would be the potential for steric clashes between target and scaffold as well as between targets in a multivalent display.…”

“…accommodate peptide extensions in the loop between adjacent repeats up to as many as 25 amino acids without compromising the native structure. 32,33 We then demonstrated that we could gra a SLiM from the protein Nrf2 that recognises the oncogenic protein Keap1 onto the scaffold and that we could not only recapitulate the native binding affinity but also improve it without need of sophisticated computational modelling. Thus, by combining this modular scaffold with SLiM graing we potentially have the capacity for diverse functionalization.…”

“…Thus, by combining this modular scaffold with SLiM graing we potentially have the capacity for diverse functionalization. 33 As proof of concept, we herein construct a set of monovalent and multivalent CTPR proteins to bind and inhibit the human poly (ADP) ribose polymerase (PARP) proteins tankyrase 1 and tankyrase 2 (referred to subsequently as hTNKS). hTNKS are unique in the PARP family in having a series of ankyrin-repeat cluster (ARC) subdomains ( Fig.…”

“…Thus, by combining this modular scaffold with SLiM grafting we potentially have the capacity for diverse functionalization. 33 …”

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

“…As it has been known that heteromeric protein-protein interactions are often mediated by loops 33,34 , a loop-grafting can be a promising way to transfer PPI onto unrelated proteins. However, only a limited number of special proteins are reported so far to be capable of presenting PPI-mediating loop peptides in an intact binding-competent conformation, and the grafting sites are also limited [35][36][37] . Choosing appropriate loop peptide moieties is also challenging, because exible peptide conformation in isolation (i.e., detached from the target) is di cult to predict, and they tend to exhibit low a nity due to the entropic penalty upon binding 34 .…”